This invention relates generally to the discovery of new regulatory compounds, drugs, and/or diagnostic agents. In particular, it relates to capillary electrophoretic methods for detecting ligands binding to a known target molecule, and for determining the relative stabilities of ligand/target complexes, thereby allowing the ranking of different ligands according to their relative binding strengths to a common target. The invention encompasses methods of detecting moderate-to-strong binding ligands in mixtures that also have much higher concentrations of competing, weaker-binding ligands.
Developing screens to identify new biologically active compounds can present unique and difficult challenges, especially when screening complex materials, particularly xe2x80x9ccomplex biological materialsxe2x80x9d: any material that may have an effect in a biological system. Examples include, but are not limited to: naturally occurring samples, products or extracts; various biological preparations; chemical mixtures; libraries of pure compounds; and combinatorial libraries. Examples of major screening problems include: detecting potential hit compounds that bind to a target of interest, especially ligands present at low concentrations in screened samples; accounting for unknown components that can interfere with screening agents; and determining the relative value of screened samples for further investigative efforts. As well, high concentrations of a weak or several weak, competing binder(s) can mask the signal from a moderate-to-strong hit compound occurring at a lower concentration in the same sample.
Recently, the use of capillary electrophoresis techniques has facilitated and improved the process of screening for unknown, biologically active compounds. For example, WO 97/22000 encompasses four broad embodiments of a capillary electrophoretic screening method, as follows.
(1) In a non-competitive embodiment of WO 97/22000, a target and complex biological sample are mixed together, then an aliquot of that target/sample mixture is subjected to capillary electrophoresis (CE), and the CE migration of the target is tracked. The target""s migration pattern under these conditions are compared against a reference standard, typically the unbound target""s migration pattern in the absence of any target-binding ligand.
(2) In a non-competitive, subtractive analysis embodiment of WO 97/22000, a target and sample are mixed together and then subjected to CE. The migration pattern of this mixture is compared to the migration pattern of a sample of the complex biological material alone. Any difference between the two migration patterns suggests the presence in the sample of a hit compound that can bind to the target.
(3) One competitive binding embodiment is provided in WO 97/22000, which tracks known, charged ligand: The target is first mixed with a complex biological material sample, and then with a known, charged ligand that binds tightly to the target, to form a sample/target/known ligand mixture. This method uses an essentially equilibrium setting when incubating target and known, tight-binding ligand together, so that the known, tight-binding ligand can displace any weaker-binding hit, prior to CE. This mixture is subjected to capillary electrophoresis and the migration of the known, charged ligand is tracked. (Thus, this method is useful when the target is not easily detected during CE.) Any difference in the known, charged ligand""s migration pattern, when in the presence of both the target and a complex biological material sample, from the known ligand""s migration pattern when in the presence of the target alone, indicates the presence of a candidate, unidentified target-binding ligand in that sample.
(4) In another competitive binding embodiment of WO 97/22000, the target""s migration is tracked and the CE running buffer contains a known, weak-binding, competitive ligand. The target is mixed with a sample, and an aliquot of the mixture is subjected to CE in the presence of a known, relatively weak, target-binding xe2x80x98competitorxe2x80x99 ligand in the CE running buffer. The migration of the target is tracked during CE. The reference standard is the migration of a target plug alone in the known ligand-containing CE buffer, its migration being shifted by its weak, reversible binding to the known ligand dispersed in the CE buffer, as compared to the target""s migration alone ligand-free buffer. This competitive screening method can detect a tight-binding hit compound in a target/natural sample mixture, because the hit binds up the target for the entire CE run and prevents the target""s interaction with the known weak-binding ligand in the buffer. Therefore, the CE migration pattern of the target in the sample/target aliquot would shift back to the target""s migration position as it would be in ligand-free running buffer. This method, too, is particularly useful when the unbound target is not easily detected in ligand-free buffer during CE.
While WO 97/22000 provides useful CE screening methods, they do not completely solve the screening problems listed previously.
Therefore, there remains a need for rapid and cost-effective screening tools for discovering new bioactive compounds and potential regulatory compounds that bind to essential molecules of key metabolic pathways. Also needed is a way of prioritizing candidate ligands and samples of material for further characterization. The present invention addresses these needs, by providing: a means of detecting unknown ligands that may be candidate, new, bioactive compounds; a means of ranking screened samples detected to contain candidate hit compounds or ligands, according to their relative binding strengths and value as potential sources of regulatory and diagnostic compounds; and a means of identifying effective and valuable, strong or moderate, target-binding ligands in the presence of weaker, competitive binders. Identifying and ranking those ligand-containing samples that form the most stable complexes with the selected target, saves time and resources spent on further isolation and characterization of hit compounds. The most stable ligands are potentially more effective and valuable as therapeutic, regulatory and/or diagnostic compounds and drugs.
The present invention provides: (1) a means to screen for target-binding ligands of a desired binding strength, in complex biological and other materials and mixtures; (2) a means to screen for the ligands or hit compounds of the desired affinity, even in the presence of weak-binding ligands in mixtures; and (3) a means to rank hit compounds according to their relative affinities. All aspects of the method can be performed on samples of pure hit compounds, partially purified hit compounds, and hit compounds present in complex biological mixtures.
The present invention provides simple and rapid, capillary electrophoretic methods for screening for and ranking ligands that do not require knowledge of a particular ligand""s structure or concentration within the screened sample. In fact, it can be practiced on samples of complex materials, such as natural samples, that potentially contain one or more unidentified ligand(s) or hit compound(s) that bind to a selected target of interest. The migration of both the target and the target/hit compound complex must be detectable during capillary electrophoresis (CE). The hit compound should be detectable by its ability to alter the CE profile of the detectable target.
The present capillary electrophoretic screening methods allow one to detect hit compounds of any desired affinity or binding strength. Detection of weak, moderate and/or strong binding ligands is possible because the screened sample, once mixed and incubated with a select target, is injected into a CE capillary and electrophoresed. Electrophoretic conditions are optimized to detect hit compounds having a desired affinity for a selected target: e.g., ligands of any binding affinity (weak, moderate, or strong), only those with moderate-to-strong affinity, or only those with strong affinity for the target. The dissociation rate or stability of a particular hit compound/target complex during CE is related to the binding strength of that hit compound to a target. The complex""s stability is also affected by such electrophoretic conditions as temperature, CE run time, and, buffer composition (e.g., its ionic strength, buffer ions, cofactors).
Therefore, using certain electrophoretic conditions, the hit compound/target complex can be stabilized so that any hit compoundxe2x80x94whether weak- , moderate- , or strong-bindingxe2x80x94is detected. Alternatively, the CE conditions can be chosen to destabilize the weak hit compound/target complex, so that weak ligands will dissociate much faster from a target than will strong ligands, during CE. The latter conditions will significantly or completely reduce a weak hit compound/target complex signal, but may only slightly reduce a strong hit compound/target complex signal. For example, a weak-binding hit compound/target complex is stabilized and detected when the temperature of the capillary is maintained relatively low, near the lower limit of viable CE temperatures (e.g., at about 5-10xc2x0 C.), and the injected sample is subjected to a relatively short capillary electrophoresis (CE) run (e.g., within a range of about 0.5-5.0 minutes, preferably 1.0-2.5 minutes), prior to the detection point where the migration of any target and/or hit compound/target complex is tracked. If the CE is performed at a higher temperature, e.g., 25-45xc2x0 C., a weak-binding ligand may dissociate and be undetectable, whereas a strong ligand may remain complexed.
One embodiment of the present screening method includes a known, charged, competitive, target-binding ligand in the capillary electrophoretic running buffer. This embodiment may be used if, when a sample is screened without a competitive ligand in the buffer, a hit compound does not detectably alter the mobility of the target upon complexation. When using a competitive ligand in the buffer, the target""s CE peak is shifted upon its complexation with competitive ligand as the target migrates through the capillary, which serves as the reference CE profile. Binding of a hit compound to a target will prevent binding of the competitive ligand and thus, will change the migration pattern of the target from the reference situation.
In its ligand-ranking aspect, the method of the invention may be used to determine the relative stabilities of different ligand/target complexes during capillary electrophoresis. Specifically, the latter aspect of the present method allows one to rank various ligands or hit compounds that are found to bind to a common target of interest, according to their xe2x80x9crelative stabilityxe2x80x9dxe2x80x94i.e., their respective ability, relative to each other, to remain complexed to that target under the same CE conditions. Some parameters influencing the stability of a target/ligand complex in CE include: the ligand""s on-rate and off-rate; capillary temperature; pH of the background buffer in the capillary; and ionic strength of the background buffer. Other factors affecting the stability of a target/ligand complex will be apparent to those of ordinary skill in the art of capillary electrophoresis.
One particularly useful application of this ligand-ranking method is in drug discovery. The method is useful for detecting candidate, unidentified ligands that bind to a selected target, in pure compound libraries or complex materials, such as biological samples or synthetic chemical mixtures. As well, unknown target-binding ligands detected to be present may be ranked in terms of their relative affinities or target-binding strengths during CE, early during drug screening and isolation protocols. Ranking of ligand-containing samples helps to save time and resources spent on follow-up isolation and characterization of potential new bioactive hit compounds.
Another particular useful application of this invention is the ability to detect strong binding ligands in the presence of higher concentrations of competitive weak binding ligands. The electrophoretic conditions affect the stability of weak and strong ligand/target complexes to different degrees. Therefore, one can set the CE conditions so that weak ligand/target complexes will dissociate completely during the CE run, but strong/ligand complexes will remain present at a level above the detection limits of the detector. The ability to identify only the samples that contain strong ligands, is very important as it helps to save time and resources spent on screening and isolation of potential, new, bioactive hit compounds.